The detection of proteins at low concentrations is of paramount importance in the areas of medicine, food testing, biological research, and the detection of biowarfare agents. By far, the most common tools for measuring proteins are antibodies, which are proteins produced by the immune systems of higher animals. Tests which use antibodies (or similar binding agents) are commonly known as “immunoassays” (Gosling, 1990).
A common alternative to using the entire antibody molecule is to use just the binding regions of the antibody. A variety of antigen-binding antibody fragments are known, such as Fab fragments (Gosling, 1990), Fab′ fragments, F(ab)2 fragments, F(ab′)2 fragments, scFv fragments, etc. Fab fragments may be obtained either by cleaving a whole antibody molecule biochemically, or by synthesizing them in cell cultures using genetic engineering techniques.
Another recently developed alternative to antibodies are aptamers (Gold et al., 1995; Drolet et al., 1996). Aptamers are nucleic acids (RNA or DNA or their derivatives) which have been designed to share many of the binding characteristics of antibodies. Aptamers offer the advantages of being produced in vitro in a comparatively short time, of having a long shelf-life, being easy to modify chemically, and of potentially exhibiting better binding characteristics than antibodies. Disadvantages include high cost, and low stability in some biological fluids.
Immunoassays are often characterized as being either “heterogeneous” or “homogeneous”. There is some confusion in the field as to what these terms mean. In some cases, the term “heterogeneous” is taken to imply that the bound complexes are separated (by any means) from the unbound molecules before detection, while the term “homogeneous” implies that no separation takes place. Another common meaning for the term “heterogeneous” (that is used in this disclosure) implies that the assay makes use of both a solid and a solution phase, in which attachment of the complex to the solid phase allows the unbound molecules to be washed away (or vice versa) before detection, while the term “homogeneous” implies that the binding, separation (if any), and detection steps occur in the solution phase. In most cases, the differing definitions make little difference in how an assay is classified, as most separation or washing steps involve binding to a solid support, and thus assays involving separation or washing would be classified as heterogeneous by either definition. The major exception is separation by electrophoresis, which would cause an assay to be classified as heterogeneous according to the first set of definitions, and homogeneous according to the second set of definitions. This disclosure uses the second set of definitions.
Therefore, in this disclosure, a heterogeneous assay involves binding the antibody-target protein complex to a surface during the test procedure and then washing away any remaining unbound sample and antibody prior to measuring the amount of protein in the sample. A homogeneous assay, on the other hand, permits the sample and antibodies to be mixed together and the result determined without any binding to a surface or washing. Each approach has both well-defined benefits and limitations (Ronkainen-Matsuno, et al. 2002).
Heterogeneous assays can have excellent sensitivity and specificity and can be performed in formats that provide for very high-throughput testing systems. These formats can also be adapted to test for virtually any antigen. Limitations include slow testing times requiring relatively sophisticated instrumentation to perform in large numbers, and the added costs associated with the surface-binding and washing steps.
Homogeneous assays can be very fast, are easily adaptable to new proteins and platforms, and can be very cost effective. Limitations include potentially lower specificity and sensitivity. In addition, these formats are typically restricted to small target antigens.
Electrophoresis is a technique that, in certain cases, may be used in place of the washing of complexes that are bound to a solid support, thereby converting what is normally a heterogeneous protein assay into a homogeneous assay. Electrophoresis is commonly used to separate molecules (usually large ones such as proteins or nucleic acids) based on their size and electrical charge (Oda and Landers, 1997). Positive and negative electrodes are placed in a solution containing the molecules to be separated, and a voltage drop is applied between the electrodes. In general, positively charged molecules will migrate toward the negatively charged electrode, while negatively charged molecules will migrate toward the positively charged electrode. The speed at which the molecules migrate is directly proportional to their charge, and inversely proportional to their size. Small, highly charged molecules move faster than large, lesser charged molecules. However, densely packed molecules move faster than loosely conformed molecules, so that two molecules of the same mass and charge may migrate at different rates. Higher voltage drops cause faster migration, while higher concentrations of other charged molecules in the solution cause slower migration.
There are many variations of electrophoresis. The solution through which the molecules move may be free, usually in capillary tubes, or it may be embedded in a matrix. Common matrices include polyacrylamide gels, agarose gels, and filter paper. The matrix serves to sieve the molecules according to size, leading to better separations. The pH (acidity) of the solution affects the charge of the molecules, and may be varied (even from one end of the matrix to the other) to affect the migration rate of the molecules. The solution may include denaturing agents such as urea, which cause protein and nucleic acid molecules to unfold, so that migration rates for molecules of the same mass and charge will be identical. The sample to be electrophoresed may be prepared with a detergent such as SDS, which coats all proteins to nullify charge differences, so that migration rates depend on mass, but not on charge. However, for most assays requiring binding agents such as antibodies or aptamers, both the protein and the binding agents need to be kept close to their natural state, so that processes that change the pH or denature the proteins are not viable options.
Present methods for detecting and/or determining the concentration of molecules in solution fail to combine short assay time, high sensitivity and ability to assay larger sample volumes required to detect and/or quantify target molecules present at very low concentration. A need exists in the field for improved analytical techniques for detection and/or concentration determination of molecules.